A number of blood coagulation proteins require a post-translational, vitamin K-dependent modification for biological activity. In 1974, Stenflo et al., Nelsestuen et al. and Magnusson et al. reported that the prototype of these vitamin K-dependent proteins, prothrombin, contained the modified amino acid, .gamma.-carboxyglutamic acid (Gla). See J. Stenflo et al., Proc. Natl. Acad. Sci. USA 71:2730-2733 (1974); G. Nelsestuen et al., J. Biol. Chem. 249:6347-6350 (1974); S. Magnusson et al.. FEBS Lett. 44:189-193. (1974). Prothrombin from animals treated with the vitamin K antagonist warfarin lacked this Gla modification. It was inferred from these observations that the blood-clotting activity of the vitamin K-dependent proteins required .gamma.-carboxylation of specific glutamic acid residues. Shortly thereafter, Esmon et al. demonstrated an enzyme activity, vitamin K-dependent carboxylase (hereafter called carboxylase), capable of making this Gla modification. See C. Esmon et al., J. Biol. Chem. 250:4744-4748 (1975).
After cDNA sequences were obtained for several of the vitamin K-dependent proteins, Pan and Price compared the deduced amino-acid sequences and suggested that the propeptide consensus sequence preceding the amino terminus of the vitamin K-dependent protein was a recognition site for the carboxylase See L. Pan and P. Price, Proc. Natl. Acad. Sci. USA 82:6109-6113 (1985). This suggestion was confirmed by Knobloch and Suttie, who demonstrated the importance of the propeptide in carboxylation by showing that the synthetic propeptide sequence of human factor X stimulated the activity of the carboxylase for a small substrate (Boc-Glu-Glu-Leu-OMe) in vitro. J. Knobloch and J. Suttie, J. Biol. Chem. 262:16157-16163 (1987). Jorgensen extended this observation by showing that factor IX with its propeptide deleted was not carboxylated. M. Jorgensen et al., Cell 48:185-191 (1987).
In spite of its importance, the carboxylase has not been previously purified. Purification of 400-fold has been reported. See J.-M. Girardot, J. Biol. Chem. 257:15008-15011 (1982). Comparison of Girardot's results to later purifications is complicated because, in our hands, ammonium sulfate and the propeptide stimulate the incorporation of CO.sub.2 into the synthetic peptide substrate FLEEL by 13-fold. If one corrects for the lack of ammonium sulfate and propeptide in Girardot's assay mix, then he achieved a specific activity of 1.1.times.10.sup.7 cpm/mg/hr. Soute et al. demonstrated that an immobilized factor X antibody would bind the carboxylase, presumably through a factor X precursor-carboxylase complex, and that the bound carboxylase retained its activity for the synthetic peptide substrate FLEEL. See B. Soute et al., Biochem, Biophys. Acta. 676:101-107 (1981). Harbeck et al. extended this method by eluting the carboxylase from a prothrombin antibody column with a synthetic propeptide achieving a 500-fold purification and a final specific activity of 6.6.times.10.sup.6 cpm/mg/hr. See M. Harbeck et al., Thromb. Res. 56:317-323 (1989). Hubbard et al. reported the purification of the carboxylase to homogeneity using a synthetic propeptide sequence as an affinity ligand. See B. Hubbard et al., Proc. Natl. Acad. Sci. USA 86:6893-6897 (1989). However, the reported final specific activity, 1.3.times.10.sup.7 cpm/mg/hr, was still not significantly different than that reported by Girardot. Numerous studies with the crude carboxylase have yielded important information about its properties and mode of action. See J. Suttie, Ann. Rev. Biochem. 54:459-77 (1985). It is clear, however, that for detailed mechanistic studies and physical characterization of the enzyme, purification is necessary.
We recently reported the production in E. coli of four different 59-residue peptides containing the propeptide and Gla domain of human factor IX. See S.-M. Wu et al., J. Biol. Chem. 265:13124-13129 (1990). We report here that one of these peptides, FIXQ/S (SEQ ID NO:1), is an excellent affinity ligand for purification of the carboxylase. A 7,000-fold purification of the carboxylase to approximately 80%-90% apparent purity and final specific activity of about 2.4.times.10.sup.9 cpm/mg/hr was obtained. The apparent molecular weight was 94,000 by reducing SDS-PAGE analysis.